AAV‐delivered diacylglycerol kinase DGKk achieves long‐term rescue of fragile X syndrome mouse model

A

Immunoblots and quantification of lysates from Cos‐1 cells transfected with plasmid pCI control or pCI‐HA‐∆N‐DGKk, with the indicated amount of plasmid (µg) and after 24 h incubated with puromycin (20 µg/ml for 30 min) to measure basal rates of protein synthesis. GAPDH was used as a loading control. Densitogram of puromycin incorporation is presented as change relative to pCI (0.2 µg) condition.

B

Cells transfected with 0.5 μg pCI, pCI‐HA‐FL‐mDGKk, or pCI‐HA‐∆N‐mDGKk plasmids were serum starved for 24 h and then incubated with 10% FCS for the indicated time period. Cells were then collected and immunoblotted with HA (DGKk), p‐mTOR, mTOR, p‐EIF4E, or EIF4E antibodies. Blots were normalized for GAPDH and then phospho/non‐phospho ratio was calculated. Upper panel: mDGKk expression. Middle panel: m‐TOR phosphorylation at Ser‐2448. Bottom panel: p‐eIF4E phosphorylation at Ser‐209. Significance was determined by 2‐way ANOVA compared to pCI control (n = 3).

C–F

Quantification of caspase 3/7 activity (C), release of lactate dehydrogenase (LDH) (D), percentage of NeunN (E), and GFAP positive cells (F), in WT and Fmr1‐KO cortical neurons untreated (NT) or transduced at 8 DIV for 8 days with indicated titers of AAV (viral genome copies) AAVRh10‐∆N‐DGKk or AAVRh10‐FMRP by immunofluorescence high‐throughput cell imaging. Positive control wells were treated with apoptotic inducer staurosporine (STS) at 0.1 and 1 µM for 6 h.

Figure EV2. ∆N‐DGKk expression modulates mTOR and EIF4E signaling without significant toxicity.